There exist fundamental gaps in the knowledge base regarding the final steps of molybdenum cofactor (Moco) biosynthesis and sulfuration, how the Mo methionine sulfoxide reductase utilizes the pyranopterin dithiolene component (MPT) of the cofactor in catalysis, and the role of non-MPT ligands in the catalytic cycles of dimethylsulfoxide reductase family enzymes. Our long-term goal is to understand geometric and electronic structure contributions to pyranopterin molybdenum enzyme reactivity and function in order to provide a positive impact on the quality of human health. Our primary objectives are to determine critical Moco maturation, transport and sulfuration steps, define the mechanism of MsrP and the role of the MPT in Msr mediated catalysis, and understand the electronic structure of key paramagnetic intermediates in DMSOR family enzymes using a combined spectroscopic approach augmented by detailed bonding, spectroscopic, and reaction coordinate calculations. The central hypothesis is that specific geometric and electronic structure modifications of protein- bound Moco define the unique reactions catalyzed. The rationale for this research is that a comprehensive understanding of Moco maturation and transport, the complex interplay between Mo the MPT in Msr catalysis, and the nature of paramagnetic DMSOR family intermediates will provide new insights into disease states and have a positive impact on human health. We will test our central hypothesis in order to accomplish the objectives of this proposal through the successful pursuit of three Specific Aims 1) Understand Moco maturation, transport, and sulfuration, 2) Determine electronic structure contributions to MsrP catalysis, 3) Determine the electronic and geometric structure of paramagnetic intermediates in DMSOR family enzymes. The proposed research is innovative in its approach because we have integrated structural, multicomponent spectroscopic, and computational investigations on models and enzymes to address critical questions concerning the final stages of Moco biosynthesis and sulfuration, Mo catalyzed repair mechanisms for oxidatively damaged proteins, and the synergistic interactions between MPT and amino acid ligation in pyranopterin Mo enzyme catalysis. This has led to new insight into long-standing questions in the molybdoenzyme field, effectively opening new horizons for future work in this area. The proposed research is significant since it will impact and advance our understanding of molybdate insertion and post-translational sulfuration processes, sulfur and Moco trafficking, molybdoenzyme mediated rescue of oxidatively damaged proteins, and the roles of amino acid and pyranopterin dithiolene ligands in molybdoenzyme catalysis.